Primary open angle glaucoma is a major blinding disease, with elevated intraocular pressure (IOP) as a key risk factor. Cells of the trabecular meshwork (TM) are responsible for maintaining IOP homeostasis. In glaucoma, reduced TM cellularity due to genetic and/or environmental factors may compromise IOP homeostatic capabilities. Current treatments aid many, but not all patients respond appropriately to them, or respond only for a limited time. Adding TM-like cells to replenish the remaining TM cells may facilitate restoration of function. We propose to evaluate two candidate stem cell types as replacements for absent/diseased TM cells. These two potentially autologous cell types, human mesenchymal stem cells (hMSC), and induced pluripotent stem cells (iPS cells), have been used to repopulate other tissues. The purpose of these replacements would be to restore normal IOP homeostasis. In this proposal, the central hypothesis is that one or both stem cell types can be induced to grow, expand, and replace diseased TM cells. We contend that they will adopt many or all of the structural, physiological, and biochemical, and functional attributes of TM in the proper microenvironment. With a multi- pronged approach, we will investigate the therapeutic potential of both stem cell types in the model ocular perfusion system by transplanting them as substitutes for diseased TM cells. In Aim #1 we will compare TM cell biomarkers to those of hMSC, iPS cells, and the differentiated stem cells. By following the progress of these markers, we will test the working hypothesis that we can use them to assess the similarities of the two stem cell types to TM cells. By culturing with aqueous humor, ECM components, different media preparations, and/or other agents, we will alter the local milieu of the stem cells. With the markers as a partial guide, differentiation of the stem cells to TM-like cells will be tracked by immunohistochemistry, confocal microscopy, qRT-PCR, and Western immunoblots. Using this approach, we can determine the best stem cell candidate for differentiation and for regenerative therapy in TM. In Aim #2 we will evaluate the functionality of transplanted cells from undifferentiated and differentiated hMSC and iPS cells. Human eyes will be experimentally partially denuded of TM cells, to test the working hypothesis that we can mimic the decreased TM cellularity of the glaucomatous eye, and then assess function of transplanted replacement cells in a model ocular perfusion system. Replacement cells will be analyzed for the capacity to restore TM-like IOP homeostasis and for phagocytotic digestion of debris. This approach has considerable promise as a novel inroad for glaucoma patients to an era of regenerative medicine.